Behavioral and Molecular Responses to Exogenous Cannabinoids During Pentylenetetrazol-Induced Convulsions in Male and Female Rats.

Epilepsy is a disabling, chronic brain disease,affecting ~1% of the World's population, characterized by recurrent seizures (sudden, uncontrolled brain activity), which may manifest with motor symptoms (e.g., convulsions) or non-motor symptoms. Temporal lobe epilepsies (TLE) compromising the hippocampus are the most common form of focal epilepsies. Resistance in ~1/3 of epileptic patients to the first line of treatment, i.e., antiepileptic drugs (AEDs), has been an important motivation to seek alternative treatments. Among these, the plant Cannabis sativa (commonly known as marihuana) or compounds extracted from it (cannabinoids) have gained widespread popularity. Moreover, sex differences have been proposed in epilepsy syndromes and in cannabinoid action. In the hippocampus, cannabinoids interact with the CB1R receptor whose membrane levels are regulated by ß-Arrestin2, a protein that promotes its endocytosis and causes its downregulation. In this article, we evaluate the modulatory role of WIN 55,212-2 (WIN), a synthetic exogenous cannabinoid on behavioral convulsions and on the levels of CB1R and ß-Arrestin2 in female and male adolescent rats after a single injection of the proconvulsant pentylenetetrazol (PTZ). As epilepsies can have a considerable impact on synaptic proteins that regulate neuronal toxicity, plasticity, and cognition, we also measured the levels of key proteins markers of excitatory synapses, in order to examine whether exogenous cannabinoids may prevent such pathologic changes after acute seizures. We found that the exogenous administration of WIN prevented convulsions of medium severity in females and males and increased the levels of phosphorylated CaMKII in the hippocampus. Furthermore, we observed a higher degree of colocalization between CB1R and ß-Arrestin2 in the granule cell layer.

Synthetic structural modifications of neurosteroid pregnanolone sulfate: Assessment of neuroprotective effects in vivo.

Neuroactive steroid 20-oxo-5ß-pregnan-3α-yl L-glutamyl 1-ester (PA-Glu), a synthetic analogue of naturally occurring 20-oxo-5ß-pregnan-3α-yl sulfate (pregnanolone sulfate, PA-S), inhibits N-methyl-D-aspartate (NMDA) receptors and possesses neuroprotective properties and minimal adverse effects. Herein, we report in vivo effects of new structural modifications of the PA-S molecule: a nonpolar modification of the steroid D-ring (5ß-androstan-3α-yl L-glutamyl 1-ester, AND-Glu), attachment of a positively charged group to C3 (20-oxo-5ß-pregnan-3α-yl L-argininate dihydrochloride salt, PA-Arg) and their combination (5ß-androstan-3α-yl L-argininate dihydrochloride salt, AND-Arg). The first aim of this study was to determine the structure-activity relationship for neuroprotective effects in a model of excitotoxic hippocampal damage in rats, based on its behavioral correlate in Carousel maze. The second aim was to explore side effects of neuroprotective steroids on motor functions, anxiety (elevated plus maze) and locomotor activity (open field) and the effect of their high doses in mice. The neuroprotective properties of PA-Glu and AND-Glu were proven, with the effect of the latter appearing to be more pronounced. In contrast, neuroprotective efficacy failed when positively charged molecules (PA-Arg, AND-Arg) were used. AND-Glu and PA-Glu at the neuroprotective dose (1 mg/kg) did not unfavorably influence motor functions of intact mice. Moreover, anxiolytic effects of AND-Glu and PA-Glu were ascertained. These findings corroborate the value of research of steroidal inhibitors of NMDA receptors as potential neuroprotectants with slight anxiolytic effect and devoid of behavioral adverse effects. Taken together, the results suggest the benefit of the nonpolar D-ring modification, but not of the attachment of a positively charged group to C3.

Aberrant expression of PAR bZIP transcription factors is associated with epileptogenesis, focus on hepatic leukemia factor.

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.

Contribution of early Alzheimer's disease-related pathophysiology to the development of acquired epilepsy.

Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAß mice whether AD-like pathology renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAß mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid-induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAß mice compared to wild-type littermates following IHK-induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK-induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aß species by intracerebroventricular Aß-specific antibody application mitigated the hyperexcitable phenotype of ArcticAß mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aß species causing widespread changes in synaptic function.

Rat intra-hippocampal NMDA infusion induces cell-specific damage and changes in expression of NMDA and GABAA receptor subunits.

Excessive stimulation of NMDA receptors with glutamate or other potent agonists such as NMDA leads to excitotoxicity and neural injury. In this study, we aimed to provide insight into an animal model of brain excitotoxic damage; single unilateral infusion of NMDA at mild dose into the hippocampal formation. NMDA infusion induced chronic, focal neurodegeneration in the proximity of the injection site. The lesion was accompanied by severe and progressive neuroinflammation and affected preferentially principal neurons while sparing GABAergic interneurons. Furthermore, the unilateral lesion did not cause significant impairment of spatial learning abilities. Finally, GluN1 and GluN2B subunits of NMDA receptor were significantly upregulated up to 3 days after the NMDA infusion, while GABAA α5 subunit was downregulated at 30 days after the lesion. Taken together, a single infusion of NMDA into the hippocampal formation represents an animal model of excitotoxicity-induced chronic neurodegeneration of principal neurons accompanied by severe neuroinflammation and subunit specific changes in NMDA and GABAA receptors.

The effect of psilocin on memory acquisition, retrieval, and consolidation in the rat.

The involvement of the serotonin system in the pathophysiology of schizophrenia has been elucidated by experiments with hallucinogens. Application of a hallucinogen to humans leads to changes in perception, cognition, emotions, and induction of psychotic-like symptoms that resemble symptoms of schizophrenia. In rodent studies, their acute administration affects sensorimotor gating, locomotor activity, social behavior, and cognition including working memory, the phenotypes are considered as an animal model of schizophrenia. The complexity and singularity of human cognition raises questions about the validity of animal models utilizing agonists of 5-HT2A receptors. The present study thus investigated the effect of psilocin on memory acquisition, reinforced retrieval, and memory consolidation in rats. Psilocin is a main metabolite of psilocybin acting as an agonist at 5-HT2A receptors with a contribution of 5-HT2C and 5-HT1A receptors. First, we tested the effect of psilocin on the acquisition of a Carousel maze, a spatial task requiring navigation using distal cues, attention, and cognitive coordination. Psilocin significantly impaired the acquisition of the Carousel maze at both doses (1 and 4 mg/kg). The higher dose of psilocin blocked the learning processes even in an additional session when the rats received only saline. Next, we examined the effect of psilocin on reinforced retrieval and consolidation in the Morris water maze (MWM). The dose of 4 mg/kg disrupted reinforced retrieval in the MWM. However, the application of a lower dose was without any significant effect. Finally, neither the low nor high dose of psilocin injected post-training caused a deficit in memory consolidation in the MWM. Taken together, the psilocin dose dependently impaired the acquisition of the Carousel maze and reinforced retrieval in MWM; however, it had no effect on memory consolidation.

Sex differences in methamphetamine pharmacokinetics in adult rats and its transfer to pups through the placental membrane and breast milk.

BACKGROUND: Methamphetamine (METH) abuse is a growing health problem worldwide, and METH use during pregnancy not only endangers the mother's health but also the developing fetus. To provide better insight into these risks, we performed the following experiments. METHOD: First, we investigated how sex influences the pharmacokinetics of METH and amphetamine (AMP) in male and female rats. Subsequently, we simulated chronic exposure of prenatal infants to METH abuse by investigating brain and plasma levels of METH and AMP in dams and pups. Finally, we modeled chronic exposure of infants to METH via breast milk and investigated sex differences in pups with regard to drug levels and possible sensitization effect of chronic prenatal METH co-treatment. RESULTS: We observed significantly higher levels of METH and AMP in the plasma and brain of female rats compared to males. Additionally, brain concentrations of METH and AMP in pups exposed to METH prenatally were equivalent to 62.13% and 37.78% relative to dam, respectively. Plasma concentrations of AMP where equivalent to 100% of the concentration in dams, while METH was equivalent to only 36.98%. Finally, we did not observe a significant effect relative to sex with regard to METH/AMP levels or sensitization effects linked to prenatal METH exposure. CONCLUSION: We demonstrated that female rats display higher levels of METH and AMP, thus indicating a greater risk of addiction and toxicity. Furthermore, our data show that pups are exposed to both METH and AMP following dam exposure.

Behavioral, neurochemical and pharmaco-EEG profiles of the psychedelic drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in rats.

RATIONALE AND OBJECTIVES: Behavioral, neurochemical and pharmaco-EEG profiles of a new synthetic drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in rats were examined. MATERIALS AND METHODS: Locomotor effects, prepulse inhibition (PPI) of acoustic startle reaction (ASR), dopamine and its metabolite levels in nucleus accumbens (NAc), EEG power spectra and coherence in freely moving rats were analysed. Amphetamine was used as a reference compound. RESULTS: 2C-B had a biphasic effect on locomotion with initial inhibitory followed by excitatory effect; amphetamine induced only hyperlocomotion. Both drugs induced deficits in the PPI; however they had opposite effects on ASR. 2C-B increased dopamine but decreased 3,4-dihydroxyphenylacetic acid (DOPAC) in the NAc. Low doses of 2C-B induced a decrease in EEG power spectra and coherence. On the contrary, high dose of 2C-B 50 mg/kg had a temporally biphasic effect with an initial decrease followed by an increase in EEG power; decrease as well as increase in EEG coherence was observed. Amphetamine mainly induced an increase in EEG power and coherence in theta and alpha bands. Increases in the theta and alpha power and coherence in 2C-B and amphetamine were temporally linked to an increase in locomotor activity and DA levels in NAc. CONCLUSIONS: 2C-B is a centrally active compound similar to other hallucinogens, entactogens and stimulants. Increased dopamine and decreased DOPAC in the NAc may reflect its psychotomimetic and addictive potential and monoaminoxidase inhibition. Alterations in brain functional connectivity reflected the behavioral and neurochemical changes produced by the drug; a correlation between EEG changes and locomotor behavior was observed.

3α5ß-Pregnanolone glutamate, a use-dependent NMDA antagonist, reversed spatial learning deficit in an animal model of schizophrenia.

Neuroactive steroids modulate receptors for neurotransmitters in the brain and thus might be efficacious in the treatment of various diseases of the central nervous system such as schizophrenia. We have designed and synthetized a novel use-dependent NMDA receptor antagonist 3α5ß-pregnanolone glutamate (3α5ß-P-Glu). In this study, we evaluate procognitive properties of 3α5ß-P-Glu in an animal model of schizophrenia induced by systemic application of MK-801. The procognitive properties were evaluated using active place avoidance on a rotating arena (Carousel maze). We evaluated effects of 3α5ß-P-Glu on the avoidance, on locomotor activity, and anxiety. 3α5ß-P-Glu alone altered neither spatial learning nor locomotor activity in control animals. In the model animals, 3α5ß-P-Glu reversed the MK-801-induced cognitive deficit without reducing hyperlocomotion. The highest dose of 3α5ß-P-Glu also showed anxiolytic properties. Taken together, 3α5ß-P-Glu may participate in the restoration of normal brain functioning and these results may facilitate the development of new promising drugs improving cognitive functioning in schizophrenia.

Monitoring of dopamine and its metabolites in brain microdialysates: method combining freeze-drying with liquid chromatography-tandem mass spectrometry.

A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus.

Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-d-aspartate receptor 3α5ß-pregnanolone glutamate.

Preclinical studies have demonstrated a considerable role for N-methyl-d-aspartate (NMDA) receptors in excitotoxicity and the concurrent neuroprotective effect of NMDA receptor antagonists. Because NMDA receptors are one of the most widespread receptors in the central nervous system, application of their antagonist often leads to serious side effects ranging from motor impairment to induction of schizophrenic-like psychosis. Therefore, we have initiated development and testing of a novel synthetic NMDA receptor antagonist derived from naturally occurring neurosteroids. 20-oxo-5ß-pregnan-3α-yl-l-glutamyl-1-ester (3α5ßP-Glu) is a novel synthetic steroidal inhibitor of the NMDA receptor. Our results show that 3α5ßP-Glu preferentially inhibits tonically activated NMDA receptors, is able to cross the blood brain barrier, does not induce psychotomimetic symptoms (such as hyperlocomotion and sensorimotor gating deficit) and reduced an excitotoxic damage of brain tissue and subsequent behavioural impairment in rats. In particular, 3α5ßP-Glu significantly ameliorated neuronal damage in the dentate gyrus and subiculum, and improved behavioural performance in active allothetic place avoidance tasks (AAPA, also known as the carousel maze) after bilateral NMDA-induced lesions to the hippocampi. These findings provide a possible new therapeutic approach for the treatment of diseases induced by NMDA receptor overactivation.

Prenatal methamphetamine exposure affects the mesolimbic dopaminergic system and behavior in adult offspring.

Methamphetamine is a commonly abused psychostimulant that causes addiction and is often abused by pregnant women. Acute or chronic administration of methamphetamine elevates the levels of the extracellular monoamine neurotransmitters, such as dopamine. The aim of the present study was to show whether prenatal exposure to methamphetamine (5mg/kg, entire gestation) or saline in Wistar rats induces changes in dopamine levels and its metabolites in the nucleus accumbens, and in behavior (locomotor activity, rearing, and immobility) after the administration of a challenge dose of methamphetamine (1mg/kg) or saline in male offspring. We found that adult offspring prenatally exposed to methamphetamine had higher basal levels of dopamine (about 288%), dihydroxyphenylacetic acid (about 67%) and homovanillic acid (about 74%) in nucleus accumbens. An increased basal level of dopamine corresponds to lower basal immobility in offspring prenatally exposed to methamphetamine. The acute injection of methamphetamine in adulthood increased the level of dopamine in the nucleus accumbens, which is related to an increase of locomotion and rearing (exploration). In addition, prenatally methamphetamine-exposed rats showed higher response to the challenge dose of methamphetamine, when compared to prenatally saline-exposed rats. In conclusion, rats exposed to methamphetamine in utero have shown changes in the mesolimbic dopaminergic system and were more sensitive to the administration of the acute dose of methamphetamine in adulthood.

Manipulation of D2 receptors with quinpirole and sulpiride affects locomotor activity before spatial behavior of rats in an active place avoidance task.

Dopamine-mediated neurotransmission is widely studied with respect to motivation, motor activity and cognitive processes. The aim of the present study was to evaluate the role of D2 receptors in the behavior of rats in the active allothetic place avoidance (AAPA) task. D2 receptor agonist quinpirole and antagonist sulpiride were administered intraperitoneally 20min prior to behavioral testing. Administration of quinpirole led to dose-dependent increase of locomotion; the spatial efficiency was spared across the dose range studied (0.05-1.0mg/kg). In contrast, sulpiride decreased locomotor activity at a dose not influencing spatial efficiency (60mg/kg); the highest dose of sulpiride (100mg/kg) caused a deficit in both locomotor and spatial behaviors. The results suggest a relatively lesser importance of D2 receptors for spatial efficiency in the AAPA task, with a predominant influence of D2 receptor ligands on motor activity.